Volume 9, Issue 8, Pages 1168-1182 (August 2016) Identification of Regulatory DNA Elements Using Genome-wide Mapping of DNase I Hypersensitive Sites during Tomato Fruit Development  Zhengkun Qiu, Ren Li, Shuaibin Zhang, Ketao Wang, Meng Xu, Jiayang Li, Yongchen Du, Hong Yu, Xia Cui  Molecular Plant  Volume 9, Issue 8, Pages 1168-1182 (August 2016) DOI: 10.1016/j.molp.2016.05.013 Copyright © 2016 The Author Terms and Conditions

Figure 1 Distribution of DNase I Hypersensitive Sites. (A) Genome-wide distribution of DNase I hypersensitive sites (DHSs) along 12 tomato chromosomes. From outside to inside, the circles represent the chromosome, gene density, DHS abundance in 20-DPA fruits, and DHS abundance in break-stage fruits. (B) Distribution of DHSs in different gene regions. (C) DHS length in different gene regions. Different letters above the columns indicate statistically significant differences between groups (Tukey's honestly significant difference test, P < 0.05). Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 2 Expression Level of Genes Associated with DHSs and H3K4me3 Modification. (A) Heatmap of percentage of genes contained one DHS or multiple DHSs in their proximal promoter with different expression levels. The genes were divided into six groups based on RPKM value: 0–1, 1–2, 2–5, 5–10, 10–30, and >30. (B) Heatmaps of DHSs around the TSS region of genes at 20 DPA and break stage. The genes containing only one DHS were used for analysis. All genes were sorted by the length of the DHSs located from −1000 to 100 bp relative to the TSS and then divided into three equal groups according to length of the DHSs: the top includes the longest 33% and the bottom the shortest 33%, with the remainder in between. (C) Percentage of genes with different expression levels in the three groups categorized in (B). The genes contained only one DHS (upper panel) and multiple DHSs (lower panel) in their proximal promoter sorted by the length of DHSs and then divided into three equal groups (top, middle, and bottom) based on the length of DHSs. Green, dark blue, yellow, gray, orange, and light blue indicate genes with RPKM values >30, 10–30, 5–10, 2–5, 1–2, and <1, respectively. (D) Percentage of genes with different expression levels associated with DHSs, H3K4me3, or both (i.e., a concurrence of DHSs and H3K4me3). Expressed genes with RPKM values greater than 0.1 were divided into 10 equal groups based on their expression levels from lowest to highest. Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 3 Changes in DNase Accessibility during Tomato Fruit Development. (A) Visualization of DHSs in 20-DPA and break-stage fruits within a region on chromosome 9. (B) Specific and common DHSs in 20-DPA and break-stage fruits. Overlapping DHSs (a minimum of 1 bp) between these two stages were merged into a single DHS. (C) Visualization of specific DHSs combined with RNA-seq read densities at three gene loci in 20-DPAfruit. (D) Visualization of specific DHSs combined with RNA-seq read densities at three gene loci in break-stage fruit. (E) Visualization of a common DHS and RNA-seq read densities at PSY1 in 20-DPA and break-stage fruits. (F) Heatmap of expressions of IAA6, IAA11, Solyc02g067380, RIN, NOR, Solyc01g006540, and PSY1 in four fruit developmental stages. Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 4 Gene-Distal DHSs Marked by H3K4me3 Overlapped with Long Non-coding RNA Genes. (A) Overlapped peaks between H3K4me3 and DHSs. Only H3K4me3 peaks or DHSs located in intergenic regions (>1 kb downstream of TTS and >3 kb upstream of TSS) were considered to be gene-distal DHSs. The others were considered to be gene-proximal DHSs or peaks. (B) Histone modification associated with gene-distal, permutated gene-distal, and promoter DHSs. The DHSs located from −1000 to 100 bp relative to TSS were considered to be promoter DHSs. (C) Common and stage-specific gene-distal DHSs marked by H3K4me3. The overlapped peaks (with a minimum of 1 bp) in the two developmental stages were merged to a new peak, which was considered to be a common peak. (D) Number of real and permutated common or stage-specific gene-distal DHSs overlapped with lncRNA genes. The permutated gene-distal DHSs were developed from randomly selected intergenic sequences (number and length of the intergenic sequences are the same as the specific or common gene-distal DHSs). This simulation was performed 1000 times. (E) Example of motif found in both break-stage-specific and common gene-distal DHSs. Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 5 Genes Regulated by Stage-Specific DHSs. (A) Number of genes associated with specific and common DHSs in 20-DPA and break-stage fruits within −1000 to 100 bp relative to the TSS. (B) Venn diagram showing the number of shared genes between those associated with specific DHSs in 20-DPA and break-stage fruits. (C) Visualization of specific DHSs in 20-DPA and break-stage fruits at Solyc04g007000. (D and E) Number of stage-specific and shared genes overlapped with RIN-associated genes (D) and potential RIN target genes (E). (F) The significance of overlap between stage-specific or shared genes and RIN-associated genes (left) or potential RIN target genes (right). Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 6 DNase I Hypersensitivity Identifies Regulatory Elements in Fruit Development and Ripening. (A) Expression profiles of the 11 279 differentially expressed genes (DEGs) at different development stages. (B) The six co-expression clusters of all DEGs. The yaxis indicates the normalized RPKM calculated as RPKM/(RPKMmean+0.0001)−1. (C) Visualization of DHSs, cis-regulatory elements, and the expression level of ENO (cluster 1) and PG2a (cluster 3) from −1000 to 100 bp relative to the TSS. (D) Enriched motifs within stage-specific DHSs of 20-DPA and break stage from −1000 to 100 bp relative to the TSS of the genes in clusters 1 and 3. Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions

Figure 7 Coordinate Regulation of Ascorbic Acid Metabolic Genes during Tomato Fruit Development. (A) The l-ascorbic acid metabolic pathway in tomato. GPI, glucose-6-phosphate isomerase; PMI, phosphomannose isomerase; PMM, phosphomannomutase; GMP, GDP-D-mannose pyrophosphorylase; GME, GDP-mannose 3′,5′-epimerase; GGP, GDP-l-galactose-1-phosphate phosphorylase; GPP, l-galactose-1-phosphate phosphatase; GalDH, l-galactose dehydrogenase; MIOX, myo-inositol oxygenase; GLDH, l-galactono-1,4-lactone dehydrogenase; AOBP, ascorbate oxidase promoter-binding protein; AO, l-ascorbate oxidase; APX, ascorbate peroxidase; MDHAR, monodehydroascorbate reductase; DHAR, dehydroascorbate reductase. (B) The expression patterns of ascorbic acid metabolic genes. (C) Visualization of DHSs from −1000 to 100 bp relative to the TSS of three ascorbic acid metabolic genes. (D) Predicted cis-regulatory element within the region from −1000 to 100 bp relative to the TSS of ascorbic acid metabolic genes. Molecular Plant 2016 9, 1168-1182DOI: (10.1016/j.molp.2016.05.013) Copyright © 2016 The Author Terms and Conditions